CN106978402B - Hybridoma cell strain Fm7A11 and fumonisin B produced by same1Monoclonal antibodies - Google Patents
Hybridoma cell strain Fm7A11 and fumonisin B produced by same1Monoclonal antibodies Download PDFInfo
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Abstract
The invention relates to a hybridoma cell strain Fm7A11 and fumonisin B generated by the same1A monoclonal antibody. The hybridoma cell strain Fm7A11(CCTCC NO: C201636) provided by the invention can be used for preparing high-titer fumonisin B1The titer of the monoclonal antibody measured by enzyme-linked immunosorbent assay (ELISA) can reach 3.2 × 105(ii) a The invention provides fumonisin B1The monoclonal antibody has high sensitivity and good specificity and is resistant to fumonisin B150% inhibition concentration IC500.32ng/mL, and fumonisin B2，B3The cross-reactions were 4.3% and 12.8%, respectively. Mixing with Zearalenone (ZEA), T-2 toxin, Ochratoxin (OTA), and aflatoxin B1(AFB1) Vomitoxin (DON) did not cross-react. Can be used for fumonisins B1And (4) quickly measuring the content.
Description
Technical Field
The invention relates to a hybridoma cell strain Fm7A11 and fumonisin B generated by the same1A monoclonal antibody.
Background
Fumonisins are a class of mycotoxins produced primarily by fusarium moniliforme and fusarium polygamum. At present, 53 fumonisins are known, among which fumonisin B1Most commonly, the medicament has the highest toxicity, can cause the white brain softening disease of horses, the pulmonary edema syndrome of pigs, and is related to human diseases such as esophagus cancer, liver cancer, stomach cancer, congenital neural tube malformation and the like. And is harmful to animal husbandry and human health. Vohorse toxin has been declared as a class 2B carcinogen by the international agency for research on cancer and the agency for environmental protection in california. The grain most polluted by fumonisins is corn, and in addition, the fumonisins exist in foods such as millet, barley, soybeans, medicinal materials, red wine, beer and the like. The growth and toxin production of fumonisins are related to the environment, strains, varieties and agricultural production. According to FDA statistics, the pollution rate of fumonisins in countries with lower climatic temperature is higher than that in countries with warmer climatic temperature in corn and products thereof all over the world, so that the dryness and humidity of the environment and the climatic temperature have important influence on the generation of the fumonisins.
The maximum limit of fumonisins has been specified internationally in over 70 countries and regions. FB in corn products for consumption by adults as specified in the European Union1And FB2The maximum residual amount is 400 mug kg-1FB in infant food1And FB2The maximum residual amount is 200 mu gkg-1Sweden specifies a limit of 1mg/kg for fumonisins in human food, and the united states Food and Drug Administration (FDA) specifies a maximum limit of 2mg/kg for fumonisins in corn for human consumption. The pollution range of fumonisins in China is wide, so that the monoclonal antibody material capable of identifying the fumonisins can be researched, the detection sensitivity of the fumonisins in agricultural products is improved, and the method has important significance for guaranteeing the safe consumption of the agricultural products in China.
The existing detection methods of fumonisins mainly comprise two categories, one is a laboratory confirmatory detection technology and an immunological detection method based on large instruments. Another class is immunoassay assays based on antigen-antibody specific recognition. Compared with expensive detection cost and labor cost of an instrumental analysis method, the immunological detection method has the advantages of simple sample treatment, high sensitivity, strong specificity and the like, and the improvement of the affinity of the core reagent antibody is the key for improving the sensitivity of the immunological detection method.
The invention aims to solve the technical problem of providing a hybridoma cell strain Fm7A11 and fumonisin B produced by the same1Monoclonal antibodies and uses thereof.
The invention provides a hybridoma cell strain Fm7A11 which is preserved in China Center for Type Culture Collection (CCTCC) at 29/3 in 2016, wherein the preservation address is China, Wuhan university, and the preservation number is CCTCCNO: C201636. It has a gene sequence for encoding the heavy chain variable region of the monoclonal antibody against fumonisin B1 shown in SEQ ID NO. 1 in the sequence table and a gene sequence for encoding the light chain variable region of the monoclonal antibody against fumonisin B1 shown in SEQ ID NO. 2 in the sequence table.
The monoclonal antibody of fumonisin B1 is secreted by hybridoma cell strain Fm7A11 with the preservation number of CCTCC NO: C201636. The heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 3 in the sequence table; the light chain variable region has an amino acid sequence shown as SEQ ID NO. 4 in the sequence table. The fumonisin B1The monoclonal antibody can recognize fumonisin B1Of p-fumonisin B2、B3The cross-reactivity ratios of (A) were 4.3% and 12.8%.
Use of an anti-fumonisin B1 monoclonal antibody in the determination of fumonisin content.
The hybridoma cell strain Fm7A11 provided by the invention is obtained by adopting a two-step screening method, and the specific steps are as follows: adopting 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) method to synthesize artificial antigen FB1-BSA. Namely FB1Activated by EDC and coupled with BSA to obtain artificial antigen FB1-BSA. The method comprises the following specific steps: 2mg of FB1Dissolving with 2mg EDC in 500 μ L0.01 mol L-15mg of BSA was dissolved in 1000. mu.L of 0.01mol/L PBS buffer. EDC solution was first added slowly to FB1To the solution, stirred at room temperature for 10min, BSA solution was added dropwise and stirred slowly at 25 ℃ for 4 h. Complete antigen FB obtained by coupling1BSA at 0.01mol L-1Dialyzed against PBS (pH 7.4) for 72 h.
BALB/c mice are completely antigenic FB via fumonisin14-6 immunizations with BSA followed by 2 times the previous immunization dose of the complete antigen FB1BSA for the last booster immunization, cell fusion after 3 days, transferring the single cell colony to a 96-well cell culture plate by using a micropipette after 2-3 weeks of cell fusion, carrying out liquid amplification culture, carrying out the first cloning, and then screening the fused cells by an ELISA method in two steps: the method comprises the steps of firstly screening positive holes resisting fumonisins but not resisting carrier protein BSA by adopting an indirect ELISA method, secondly detecting culture solution of the positive holes screened in the first step by adopting an indirect competitive ELISA method, selecting holes with higher light absorption values and inhibition rates by adopting a fumonisin B1 standard substance as a competitive antigen, subcloning by adopting a limiting dilution method, detecting by adopting the same two-step screening method after subcloning, and finally screening to obtain a hybridoma cell strain Fm7A11 after repeating the subcloning for 4-5 times.
The invention provides fumonisin B1The preparation method of the monoclonal antibody comprises the following steps: injecting the hybridoma cell strain Fm7A11 obtained above into the abdomen of BALB/c mouse treated with Freund's incomplete adjuvant in advance, collecting ascites of the mouse, and purifying to obtain fumonisin B1A monoclonal antibody.
According to the scheme, the purification method is an octanoic acid-ammonium sulfate method, and comprises the following specific steps: filtering ascites of mice with double-layer filter paper, centrifuging at 4 deg.C and 12000r/min for more than 15min, sucking supernatant, mixing the obtained ascites supernatant with 4 times volume of acetate buffer solution, slowly adding n-octanoic acid under stirring, wherein the volume of n-octanoic acid required by each milliliter of ascites is 30-35uL, mixing at room temperature for 30-60min, and standing at 4 deg.C for more than 2 h. 12000r/min, centrifuging at 4 ℃ for more than 30min, discarding the precipitate, filtering the obtained supernatant with double-layer filter paper, adding 1/10 phosphate buffer solution with the molar concentration of 0.1mol/L and the pH of 7.4, adjusting the pH of the mixed solution to 7.4 with 2mol/L sodium hydroxide solution, slowly adding ammonium sulfate in ice bath until the final concentration of ammonium sulfate is 0.277g/mL, standing at 4 ℃ for more than 2h, then centrifuging at 12000r/min at 4 ℃ for more than 30min, discarding the supernatant, precipitating with 1/10 volume of original ascites with phosphorus with the molar concentration of 0.01mol/L, pH of 7.4And (3) resuspending the salt buffer solution, putting the salt buffer solution into a dialysis bag, dialyzing the solution for two days by using 0.01mol/LPBS, dialyzing the solution for two days by using PB, taking out the protein solution in the dialysis bag, centrifuging the protein solution, collecting supernatant, removing precipitate, pre-freezing the precipitate at-70 ℃, and putting the precipitate into a freeze dryer for freeze drying. Collecting the freeze-dried powder to obtain the purified fumonisin B1A monoclonal antibody.
The acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.01mol/L phosphate buffer solution is prepared by adding water into 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate to reach a constant volume of 100 mL; the 0.1mol/L phosphate buffer solution is prepared by adding water to a constant volume of 100mL, wherein the phosphate buffer solution is 8g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium chloride and 0.2g of potassium dihydrogen phosphate.
The invention has the beneficial effects that:
(1) the hybridoma cell strain Fm7A11 provided by the invention can be used for preparing high-valence fumonisin B1The titer of the monoclonal antibody can reach 3.2 × 10 by enzyme-linked immunosorbent assay (ELISA)5。
(2) The monoclonal antibody for resisting fumonisin B1 provided by the invention has high sensitivity and good specificity, and can resist fumonisin B150% Inhibitory Concentration (IC)50) 0.32ng/kg, p-fumonisin B2、B3The cross-reactivity ratios of (A) were 4.3% and 12.8%. The cross reaction rate of the alpha-amino acid with aflatoxin, zearalenone, T-2 toxin, ochratoxin and vomitoxin is less than 0.1 percent.
(3) The fumonisins B provided by the invention1Monoclonal antibody can be applied to fumonisin B1And (4) measuring the content.
Drawings
FIG. 1 shows fumonisins B provided by the present invention1Schematic diagram of fumonisin immunochromatographic test strip prepared by monoclonal antibody.
FIG. 2 shows the use of the anti-fumonisins B provided by the invention in example 51Fumonisin B prepared by monoclonal antibody1And (3) a result judgment diagram of a detection sample of the immunochromatographic test paper.
In the figure: 1, a water absorption pad; 2 detecting the pad; 3, controlling the quality; 4, detecting lines; 5, gold mark pad; 6 a sample pad; 7, paper boards; 8 control test strips; 9 test strip.
Detailed Description
Example 1: screening of hybridoma cell line Fm7A11
1. Antigen synthesis and animal immunization
Purchase of commercial fumonisin B1The standard substance is subjected to complete antigen synthesis, and the specific synthesis step is that 2mg of FB is added1Dissolving the standard powder and 2mg EDC in 500 μ L of 0.01mol/LPBS solution to obtain EDC solution and FB1Solution 4mg/mL (0.01 mol/LPBS solution) EDC solution was added dropwise to the dissolved FB1The solution was gently stirred at room temperature for 10 minutes. A5 mg/mL (0.01 mol/LPBS solution) BSA solution was added dropwise to the above mixture, and the reaction was stirred at room temperature for 4 hours. Dialyzed for 3 days. Finally, the conventional ultraviolet scanning method is used for identification, and the identification result shows that FB1The BSA complete antigen was successfully prepared.
6 BALB/c mice of 6 weeks old were purchased and immunized with fumonisin complete antigen FB synthesized in laboratory1-BSA. For the first immunization, fumonisin complete antigen is emulsified with equal volume of Freund complete adjuvant and then injected into the neck and back of a mouse at multiple points. The second immunization was performed 3 weeks later, emulsified with equal volume of fumonisin complete antigen in Freund's incomplete adjuvant, and injected subcutaneously at multiple points in the back of the mouse neck. The third and fourth immunizations are separated from the last immunization by two weeks, respectively, and the immunization mode is the same as the second immunization. The four immunization doses were identical and were only 100. mu.g/mouse. And on the 7 th day after the third immunization, blood is collected from the tail vein of the mouse, serum is separated, the titer of the mouse serum is monitored by adopting an indirect ELISA method, the sensitivity of the mouse serum is measured by adopting an indirect competitive ELISA method, the mouse corresponding to the serum with higher titer and sensitivity is selected for the last boosting immunization, and the immunization dose is 2 times of the previous dose.
2. Cell fusion
After 3 days of the last boosting immunization, 50 percent (weight percentage) of polyethylene glycol (PEG) with the molecular weight of 1450 is adopted as a fusion agent to carry out cell fusion according to a conventional method, and the specific steps are as follows: the mice to be fused were killed by decapitation under sterile conditions, spleen cells were isolated, and the ratio of mouse-derived myeloma cells SP2/0 to 5: 1, washing the mixed cells with RPMI-1640 basic culture medium, 1200rpm, and centrifuging for 5 min. Discarding the supernatant, draining, adding 1mL of PEG, fusing for 1 minute, slowly adding RPMI-1640 basic culture solution, centrifuging, discarding the supernatant to obtain a precipitate as fused cells, re-suspending with 20mL of complete culture medium, adding the suspended cells into 80mL of semisolid culture medium, uniformly mixing, adding onto a 6-well cell culture plate, culturing at 2 mL/well, and placing in a carbon dioxide incubator at 37 ℃.
The complete cell culture medium containing 1% HAT contains 20% (volume percent) of fetal calf serum, 75% (volume percent) of RPMI-1640 basic culture solution, 1% (weight percent) of L-glutamine, 1% (volume percent) of HEPES, 1% (volume percent) of diabody (10000 units per milliliter of penicillin and 10000 micrograms per milliliter of streptomycin), 2% (volume percent) of growth factor (HFCS) and 1% (weight percent) of hypoxanthine-aminopterine-thymidine, namely HAT and methylcellulose, which are purchased from sigma-Aldrich company.
3. Screening and cloning of cell lines
And (3) picking out the clone from the culture medium by using a micropipette when the cell colony grows to be visible by naked eyes 2-3 weeks after the cells are fused, transferring the clone to a 96-hole cell culture plate, culturing by using HAT liquid, and sucking culture supernatant for detection when the cells grow to the bottom of 2/3 holes. Adopting a two-step screening method, and adopting an indirect ELISA method in the first step to screen out positive holes which resist fumonisin but not resist carrier protein BSA; the second step adopts indirect competition ELISA method to detect the positive hole screened in the first step, and fumonisin B is used1As the competitor, a well having a high absorbance and a high sensitivity (a high absorbance means that the final measurement value of a positive control well, which is a well having a competitor of 0, is high, and a high sensitivity means that the concentration of the competitor at an inhibition of 50% is also IC50Smaller value), carrying out subcloning by using a limiting dilution method, carrying out detection by using the same two-step method after subcloning, and repeating the subcloning for 4-5 times to obtain the hybridoma cell strain Fm7A 11.
Example 2: anti-fumonisins B1Monoclonal antibody hybridoma cell strain Fm7A11 antibody variable region sequence determination
(1) Extracting total RNA: extracting total RNA capable of generating hybridoma cell strain Fm7A11 by adopting a total RNA extraction kit of Tiangen company according to an instruction;
(2) synthesis of cDNA: taking the total RNA obtained in the step 1 as a template, and oligo (dT)15 as a primer according to SuperScriptTM-2II reverse transcriptase instructions for reverse transcription to synthesize first strand cDNA; primer oligo (dT)15 was purchased from Invitrogen;
(3) the PCR method is to design primers according to conserved sites of a mouse antibody gene sequence in GENBANK, amplify the heavy chain and light chain variable region genes of the antibody by using CDNA as a template, wherein the PCR program comprises the steps of amplifying the heavy chain and light chain variable region genes by using CDNA as a template, amplifying the genes for 30 cycles at 94 ℃, 58 ℃ for 45S and 72 ℃ for 1min, finally extending the products for 10min at 72 ℃, separating the PCR products by agarose gel electrophoresis at 1 percent (weight percent), purifying and recovering DNA fragments by using a kit, connecting the DNA fragments to a vector pMD18-T, transforming escherichia coli DH5 α competent cells, picking up positive clones, and sending the positive clones to Hippocampus Sangnie Biotech limited for sequencing, wherein the sequences of the primers are respectively 5 '-CAG GTS MAR CTG MAG GAG TCW G-3' (22mer) and 5'-CAG GGG CCA GTGGAT AGA CAG ATG GGG G-3' (28mer) of the heavy chain variable region primer, wherein S, M, R and W are merged bases, M ═ A/C, R ═ A/G, S ═ G/C, W ═ A/T, and light chain variable region primer 5'-GAC ATC AAG ATG ACC CAG TCT CCA-3' (24mer) and 5'-CCGTTT TAT TTC CAG CTT GGT CCC-3' (24 mer).
Results of the gene sequences obtained: the length of the gene sequence of the heavy chain variable region coding gene is 379bp, the sequence is shown as SEQ ID NO. 1, the heavy chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 126 amino acids, and the sequence is shown as SEQ ID NO. 3. The light chain variable region coding gene sequence has the length of 348bp and is shown in SEQ ID NO. 2, the light chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 116 amino acids, and the sequence is shown in SEQ ID NO. 4.
Example 3: anti-fumonisins B1Preparation and purification, subtype and characteristic identification of monoclonal antibody
Mixing all the materialsAnti-fumonisin B obtained in example 11The monoclonal antibody hybridoma cell strain Fm7A11 is injected into a BALB/c mouse which is treated by Freund's incomplete adjuvant in advance, ascites of the mouse is collected, and an octanoic acid-ammonium sulfate method is adopted to purify the antibody, and the concrete operation is as follows: filtering ascites of mice with double-layer filter paper, centrifuging at 4 deg.C and 12000r/min for more than 15min, sucking supernatant, mixing the obtained ascites supernatant with 4 times volume of acetate buffer solution, slowly adding n-octanoic acid under stirring, wherein the volume of n-octanoic acid required by each milliliter of ascites is 30-35 μ L, mixing at room temperature for 30-60min, and standing at 4 deg.C for more than 2 h. Centrifuging at 12000r/min at 4 deg.C for more than 30min, discarding precipitate, filtering the obtained supernatant with double-layer filter paper, 1/10 adding phosphate buffer solution with molarity of 0.1mol/L and pH of 7.4, adjusting pH of the mixture to 7.4 with 2mol/L sodium hydroxide solution, slowly adding ammonium sulfate in ice bath to final concentration of 0.277g/mL, standing at 4 deg.C for more than 2 hr, then 12000r/min, centrifuging at 4 ℃ for more than 30min, discarding the supernatant, resuspending the obtained precipitate with phosphate buffer solution with the molar concentration of 0.01mol/L, pH of 7.4 of the volume of original ascites volume 1/10, filling into a dialysis bag, dialyzing for two days with 0.01mol/LPBS, dialyzing for two days with PB again, taking out the protein solution in the dialysis bag, centrifuging, collecting the supernatant, discarding the precipitate, pre-freezing at-70 ℃, and freeze-drying in a freeze-dryer. Collecting the freeze-dried powder to obtain the purified fumonisin B1A monoclonal antibody;
the acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.01mol/L phosphate buffer solution is prepared by adding water into 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate to reach a constant volume of 100 mL; the 0.1mol/L phosphate buffer solution is prepared by adding water to a constant volume of 100mL, wherein the phosphate buffer solution is 8g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium chloride and 0.2g of potassium dihydrogen phosphate.
Method for identifying fumonisin B secreted by hybridoma cell strain Fm7A11 by using commercial subtype identification kit1The subtype of monoclonal antibody is IgG2 b.
The titer of the antibody obtained by ascites purification of the mice by the conventional non-competitive enzyme-linked immunosorbent assay (ELISA) can reach 3.2 × 105I.e. antibody dilution 3.2 × 105The solution test result is positive. Determination of its fumonisin B by conventional indirect competitive ELISA1The sensitivity was 0.32 ng/mL. P-fumonisin B2、B3The cross-reactivity ratios of (A) were 4.3% and 12.8%. The cross reaction rate of the alpha-amino acid with aflatoxin, zearalenone, T-2 toxin, ochratoxin and vomitoxin is less than 0.1 percent.
Example 4: antibody applications
The fumonisin immunochromatographic test strip is prepared from an anti-fumonisin B1 monoclonal antibody secreted by a hybridoma cell strain Fm7A11, and the preparation method comprises the following steps:
(1) preparation of absorbent pad
Cutting the absorbent paper into pieces with length of 15-20mm and width of 3.4mm to obtain absorbent pad;
(2) preparation of detection pad
Coupling substance FB of fumonisin-bovine serum albumin1OVA is prepared into coating solution with the concentration of 0.25mg/mL by using the coating buffer solution; coating the nitrocellulose membrane on a position 15mm away from the nitrocellulose membrane in a point spraying manner to obtain detection lines, wherein each centimeter of FB is required for the detection lines1-320 ng of conjugate coating of BSA, then dried at 37 ℃ for 30 min;
coating of quality control line:
coating buffer solution is used for preparing a rabbit anti-mouse polyclonal antibody into coating solution with the concentration of 0.8 mg/mL; transversely coating the test line on a nitrocellulose membrane at a position 6mm away from the test line by a dot spraying mode to obtain a quality control line, wherein the coating amount of rabbit anti-mouse polyclonal antibody required by each centimeter of the quality control line is 400ng, and then drying for 1 hour at 37 ℃;
each 10mL of the coating buffer solution contains 0.1g of bovine serum albumin, 0.08g of sodium chloride, 0.029g of disodium hydrogen phosphate dodecahydrate, 0.002g of potassium chloride and 0.002g of potassium dihydrogen phosphate;
the length of the nitrocellulose membrane is 25mm, and the width of the nitrocellulose membrane is 3.4 mm;
(3) preparation of sample pad:
cutting glass cellulose into pieces with length of 13mm and width of 3.4mm, soaking in sealing liquid, taking out, drying at 37 deg.C for 6 hr to obtain sample pad, and storing at room temperature in a dryer;
(4) preparing a gold label pad:
cutting a glass fiber membrane into a specification with the length of 10mm and the width of 3.4mm, soaking in a sealing solution, taking out, drying for 6 hours at 37 ℃, transversely spraying a nano-gold-labeled anti-fumonisin B1 monoclonal antibody solution on the dried glass fiber membrane in a dot spraying manner, wherein the required nano-gold-labeled anti-fumonisin B1 monoclonal antibody per centimeter of spraying length is 210ng, performing vacuum freeze drying for 2.5 hours, and placing in a dryer for room-temperature storage;
the confining liquid in the steps (3) and (4) is 1g of ovalbumin, 2g of sucrose, 0.02g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate, and water is added to the confining liquid to be 100mL in constant volume;
the specific labeling method of the nanogold-labeled fumonisin B1 monoclonal antibody solution comprises the following steps: measuring 50.0mL of nano gold solution with the mass concentration of 0.01%, and adjusting the pH value of the solution by 700 mu L of 0.1mol/L potassium carbonate aqueous solution; slowly adding 1.5mL of 0.1mg/mL anti-fumonisin B1 monoclonal antibody aqueous solution under the stirring state, and continuously stirring for reaction for 30 min; adding 10% bovine serum albumin aqueous solution to final mass concentration of 1%, and stirring for 30 min; standing at 4 deg.C for 2 hr, centrifuging at 3000r/min for 15min, collecting supernatant, and discarding precipitate; centrifuging the supernatant for 20min at 12000r/min, discarding the supernatant, re-suspending the precipitate with a labeled washing preservation solution to obtain 5.0mL of concentrate, and placing the concentrate in a refrigerator at 4 ℃ for later use, wherein the mass concentration of the nano-gold labeled fumonisin B1 monoclonal antibody solution is 10 mug/mL;
the particle size of the nano-gold in the nano-gold solution is 12 nm;
the 0.1mol/L potassium carbonate aqueous solution is as follows: dissolving 13.8g of potassium carbonate in pure water to reach the constant volume of 1000mL, and filtering with a 0.22-micron filter membrane to obtain the potassium carbonate solution; the 0.1mg/mL anti-fumonisin B1 monoclonal antibody water solution is prepared by dissolving 1mg anti-fumonisin B1 monoclonal antibody in 10mL pure water; the 10% bovine serum albumin water solution is obtained by dissolving 10g bovine serum albumin in 100mL pure water and filtering with a 0.22 mu m filter membrane; the marked washing and preserving fluid is as follows: 2.0g of polyethylene glycol-20000, 0.2g of sodium azide, 0.1235g of boric acid and pure water, wherein the volume of pure water is 1000mL, and the pure water is obtained by filtering through a 0.22-micron filter membrane;
(5) assembling the test strip:
sequentially adhering a water absorption pad, a detection pad, a gold mark pad and a sample pad on one surface of the paperboard from top to bottom, and overlapping and connecting adjacent pads at the joint, wherein the overlapping length is 1-3mm, thus obtaining the fumonisin immunochromatographic test strip, which is shown in figure 1.
Example 5: the fumonisin immunochromatographic test strip is applied as follows:
and (3) processing of the corn sample: grinding a 20g corn sample by using a grinder, adding 100mL of 80% (volume fraction) methanol water after grinding, stirring and reacting for 2 minutes to obtain a sample mixed solution, manually shaking the mixed solution for 3 minutes, filtering by using double-layer filter paper, collecting 2mL of filtrate, adding 4mL of pure water to dilute the filtrate, and uniformly mixing to obtain a sample detection solution. And (3) taking another blank corn sample of the known fumonisins, and carrying out the same treatment to obtain a blank corn sample detection solution.
The fumonisin test paper strip is used for detecting corn samples: sucking 100 mu L of each of the No. 1 and No. 2 corn sample detection solutions, respectively dropwise adding a sample pad of a fumonisin immunochromatographic test strip, and taking the sample pad as a detection test strip; meanwhile, 100 mu L of blank corn sample detection solution without fumonisins is dropwise added into a sample pad of another fumonisins immunochromatographic test strip to be used as a control test strip, and the result is read after 15 min.
And (3) detection results: the quality control line and the detection line of the control test strip both show red strips; referring to the figure 2-1, the quality control line of the No. 1 sample test strip shows a red line, the detection line does not develop color, the test strip is judged to be a positive result, the content of fumonisin in the sample solution to be detected is equal to or higher than 20ng/mL, and the fumonisin B in the corn sample is obtained through conversion1The content is equal to or higher than 300 ng/mL. The quality control line of the 2# sample test strip shows a red strip, but the color of the test line is lighter than that of the test line of the control test strip, so that the following judgment is made: a positive result is obtained, and the fumonisin content in the sample solution to be detected is equal to or higher than 0.5 ng-mL, and less than 20ng/mL, as shown in FIGS. 2-2, in terms of fumonisin content in corn sample # 2 equal to or greater than 7.5ng/mL, and less than 300 ng/mL.
Sequence listing
< 110 > institute of oil crops of Chinese academy of agricultural sciences
Hybridoma cell strain Fm7A11 with the purity of < 120 > and fumonisin B produced by the same1Monoclonal antibodies
＜160＞ 4
＜210＞ 1
＜211＞ 379bp
＜212＞ DNA
< 213 > mice
＜400＞ 1
caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag 50
cctgtccatc acttgcactg tctctgggct ttcattaacc agctatggtg 100
tacactgggt tcgtcaggcc ccaggaaagg gtctggagtg gctgggagta 150
atttggggtg gtggaaacac aaattataat tcggctctca tgtccagact 200
gagcatcagc aaagacaact ccaggagcca agttttctta agaatgaaca 250
gtctgcaaat tgatgacaca gccatgtact attgtgccag aggcaggatg 300
gactactggg gtcaaggaac ctcagtcacc gtctcgtcag ccaaaacgac 350
acccccatct gtctatccac tggcccctg 379
＜210＞ 1
＜211＞ 348bp
＜212＞ DNA
< 213 > mice
＜400＞ 2
gacatcaaga tgacccagtc tccatcttcc atgtatgcat ctctaggaga 50
aagagtcact atcacttgca aggcgagtca ggacattagt agctatttag 100
gctggttaca gcagaaacca gggaaatctc ctaagaccct gatctatcgt150
gcaaacacat tggtagaagg ggtcccatcc agattcagtg gcagtggatc 200
tggggaagat tattctctca ccatcagcag cctggagtat gaagatatgg 250
gaatttatta ttgtctacag tatgatgagt ttccgtacac gttcggaggg 300
gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatcc 348
＜210＞ 1
＜211＞ 126
＜212＞ PRT
< 213 > mice
＜400＞ 3
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser 15
Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Leu Ser Leu Thr 30
Ser Tyr Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 45
Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Asn Thr Asn Tyr Asn 60
Ser Ala Leu Met Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Arg 75
Ser Gln Val Phe Leu Arg Met Asn Ser Leu Gln Ile Asp Asp Thr 90
Ala Met Tyr Tyr Cys Ala Arg Gly Arg Met Asp Tyr Trp Gly Gln 105
Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser 120
Val Tyr Pro Leu Ala Pro 126
＜210＞ 1
＜211＞ 116
＜212＞ PRT
< 213 > mice
＜400＞ 4
Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu 15
Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Ser 30
Ser Tyr Leu Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Pro Lys 45
Thr Leu Ile Tyr Arg Ala Asn Thr Leu Val Glu Gly Val Pro Ser 60
Arg Phe Ser Gly Ser Gly Ser Gly Glu Asp Tyr Ser Leu Thr Ile 75
Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln 90
Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 105
Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser 116
Claims (4)
1. Hybridoma cell strain Fm7A11, characterized in that: it is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C201636.
2. Anti-fumonisins B1The monoclonal antibody secreted by the hybridoma cell strain Fm7A11 with the preservation number of CCTCC NO: C201636 as claimed in claim 1.
3. Anti-fumonisin B according to claim 21Monoclonal antibody in fumonisin B1Use in assays.
4. Anti-fumonisin B according to claim 21A method for producing a monoclonal antibody, characterized in that: injecting the hybridoma cell line Fm7A11 of claim 1 into BALB/c mouse treated with Freund's incomplete adjuvant, collecting ascites of the mouse, purifying to obtain fumonisin B1A monoclonal antibody.
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| CN201710131166.0A CN106978402B (en) | 2017-03-07 | 2017-03-07 | Hybridoma cell strain Fm7A11 and fumonisin B produced by same1Monoclonal antibodies |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429250A (en) * | 2008-12-12 | 2009-05-13 | 湖南农业大学 | Mold toxin penicillic acid monoclone antibody and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429250A (en) * | 2008-12-12 | 2009-05-13 | 湖南农业大学 | Mold toxin penicillic acid monoclone antibody and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| An ultrasensitive gray-imaging-based quantitative immunochromatographic detection method for fumonisin B1 in agricultural products.;Tang et al.;《Food Control》;20170516;333-340 * |
| Immunity Theory-Based High-Specific Monoclonal Antibody Preparation and Application of Fumonisin B1.;Li et al.;《Food Anal. Methods》;20170427;3361-3367 * |
| 伏马菌素B1单克隆抗体的制备及免疫学检测方法初步应用;姚静静 等;《畜牧兽医学报》;20161231;第47卷(第5期);摘要 * |
| 抗伏马菌素B1单链抗体的制备及其免疫学检测初步应用研究;邹龙;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20140315(第03期);B024-33 * |
| 玉米中伏马菌素免疫学快速检测方法研究;李岩松;《中国博士学位论文全文数据库》;20120515(第05期);E055-6 * |
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